Title: NAT Testing Using the Roche COBAS Amplicor

NCTVL Standard Operating Procedure (SOP)Title: PBMC Specimen Processing for PAR Immunoassay Page 1 of 12Doc. #: SOP340521 Revision: RETIRED Effective Date: 7/24/2009National Clinical Target Validation Laboratory (NCTVL)Applied Developmental DirectorateSAIC-Frederick, Inc.NCI-Frederick Cancer Research FacilityRevisionApprovalDateChange HistoryDescription Originator Approval-- 7/13/2006 New document adopted from LHTP RP JJA 10/13/2006 Format change and revision YZ JJB 9/19/2007 PBMC vial changes KL JJC 10/14/2008D 12/01/2008Merge PBMC Preparation (SOP34503) withExtraction Method (SOP34506)Updated SOP Web site, SOP title, andmoved reagent preparation to Batch Recordfor technician sign-off7/24/2009 RETIREDKGYZJJJJNOTICE:SOP340521 has been retired.• SOP340521: PBMC Specimen Processing for PAR Immunoassay, has been separatedinto 2 SOPs;o SOP340503 outlines PBMC collection and handling, ando SOP340506 outlines protein extraction from the prepared PBMCs.Processing Samples from Patients Currently on PAR Inhibitor Clinical Trials• If some of a patient’s specimens have been collected following SOP340521, collect andprocess the remaining samples for that patient following the same SOP to ensureconsistency in sample handling.• For any new patients, use the updated PBMC Specimen Handling SOP, SOP340503, tocollect and process samples for shipment to NCTVL.Please check the DCTD Biomarkers Web sitehttp://dctd.cancer.gov/ResearchResources/ResearchResources-biomarkers.htmfor the current status of SOP340503 and SOP340506

NCTVL Standard Operating Procedure (SOP)Title: PBMC Specimen Processing for PAR Immunoassay Page 2 of 12Doc. #: SOP340521 Revision: RETIRED Effective Date: 7/24/20091.0 PURPOSETo standardize the method for preparing lysates of peripheral blood mononuclear cells(PBMC) to enable quantification of poly (ADP-ribose) (PAR) levels with an Enzyme-Linked ImmunoSorbent Assay (ELISA) in pharmacodynamic (PD) studies of PARpolymerase (PARP) inhibitors.2.0 SCOPEThis procedure applies to all personnel responsible for the processing of blood frompatients participating in clinical trials of PARP inhibitors to isolate PBMCs and thenprepare extracts for the analysis of PAR levels by the PAR Immunoassay (SOP340505).3.0 ABBREVIATIONSCEB = Cell Extraction BufferCPT = Cell Preparation TubeDCTD = Division of Cancer Treatment and DiagnosisELISA = Enzyme-Linked ImmunoSorbent AssayID = IdentificationNCI = National Cancer InstitutePI = Protease InhibitorPBS = Phosphate Buffered SalinePAR = Poly (ADP-Ribose)PARP = Poly (ADP-Ribose) PolymerasePBMC = Peripheral Blood Mononuclear CellsPD = PharmacodynamicPMSF = Phenylmethanesulfonyl fluorideSOP = Standard Operating ProcedureRT = Room Temperature

NCTVL Standard Operating Procedure (SOP)Title: PBMC Specimen Processing for PAR Immunoassay Page 3 of 12Doc. #: SOP340521 Revision: RETIRED Effective Date: 7/24/20094.0 INTRODUCTIONThe PAR Immunoassay (SOP340505) has been developed to measure the impact ofPARP inhibitors on PAR levels in a variety of biospecimen types. This SOP outlines therecommended procedure for the preparation of PBMCs to ensure consistency in sampleextraction of PAR-containing macromolecules.5.0 RESPONSIBILITIES5.1 It is the responsibility of the Laboratory Supervisor/Manager to ensure that allpersonnel have documented training and certification on this SOP prior to theactual handling and processing of specimens from clinical trial patients.5.2 It is the responsibility of the Laboratory Supervisor to confirm scheduledspecimen collection time points, print all labels and data collection sheets inadvance, check documentation for accuracy, and verify that the requiredcollection tubes, supplies, and equipment are available for successful isolation andextraction of PBMCs.5.3 It is the responsibility of the Laboratory personnel to ensure timely transport andprocessing of the samples, enter and review all of the required collection andprocessing data, and archive all data sheets in the appropriate files.5.4 The Laboratory Technician responsible for the preparation and processing of thePBMCs is to follow this SOP and complete the required tasks and associateddocumentation. The Batch Record (Appendix 1) must be completed for eachexperimental run, with each page dated and initialed, and placed with the clinicalspecimen information.5.5 The responsible personnel are to check the DCTD Biomarkers Web site(http://dctd.cancer.gov/ResearchResources/ResearchResources-biomarkers.htm)to verify that the latest SOP version is being followed.6.0 MATERIALS AND EQUIPMENT REQUIRED6.1 Sorvall Legend RT centrifuge (Fisher Scientific)6.2 Sorvall Fresco centrifuge (Fisher Scientific)6.3 Ultrasonic Processor (Cole-Parmer Instruments, Model#: CP 130PB)6.4 Hemacytometer6.5 Pipettors (1000 µL, 200 µL, 20 µL) and tips6.6 Electronic pipette

NCTVL Standard Operating Procedure (SOP)Title: PBMC Specimen Processing for PAR Immunoassay Page 4 of 12Doc. #: SOP340521 Revision: RETIRED Effective Date: 7/24/20096.7 1.5-mL Sarstedt o-ring screw cap tubes (Fisher, Cat#: 72.694.005)6.8 2.0-mL Sarstedt o-ring screw cap tubes (Fisher, Cat#: 72.694.006)6.9 3-mL Falcon transfer pipette (Fisher, Cat# 13-711-6)6.10 15-mL polypropylene tubes (Fisher, Cat#: 14-959-49B or Becton Dickinson,Cat#: 352097 or 352095)6.11 Vacutainer Cell Preparation Tubes (CPT) (blue/black conventional closure;Becton Dickinson, Cat#: 362760)6.12 Plasma-Lyte A pH 7.4, USP (Baxter, NDC 0338-631703 or 0338-6317-04)6.13 Trypan Blue, 0.4%, sterile (StemCell Technologies, Cat#: 07050)6.14 Phenylmethanesulfonyl fluoride (PMSF) (Sigma, Cat#: 93482-50ML-F)6.15 Protease Inhibitor (PI) Cocktail (Sigma, Cat#: P-2714 or Roche,Cat#: 11 697 498 001)6.16 Cell Extraction Buffer (CEB; Invitrogen, Cat#: FNN0011)6.17 100% ethanol6.18 Dry ice6.19 81-cell chipboard storage boxes (Fisher, Cat#: 12-565-182)6.20 Thermoflask cooler6.21 Ice bucket6.22 -80°C freezer*If instruments and/or reagents differ from those specified above, the Laboratoryprocessing the clinical specimens must prove their comparability or equivalence to thoserecommended using the manufacturer’s specifications and experimental validation data.7.0 OPERATING PROCEDURES7.1 All reagents for an individual assay are to be prepared for use in one experimentalrun, and only in the amounts required for the specific assay. All excess reagentsare to be discarded following appropriate safety procedures. The buffers toprepare are listed in the Batch Record (Appendix 1, Section 1).7.2 Blood Collection and PBMC Preparation7.2.1 Ensure that the phlebotomist is using the recommended 4-mL BectonDickinson Vacutainer CPTs to draw the blood samples. If necessary,supply the phlebotomist with the correct CPTs. Four identical specimenlabels are to be prepared for each time point as defined in thePharmacodynamic/Correlative Study section of the clinical protocol.7.2.2 The research nurse is to notify the laboratory of scheduled PD samplecollections, preferably giving at least 24-h notice. A laboratory technicianis to arrive at the blood collection site at least 5 min ahead of the

NCTVL Standard Operating Procedure (SOP)Title: PBMC Specimen Processing for PAR Immunoassay Page 5 of 12Doc. #: SOP340521 Revision: RETIRED Effective Date: 7/24/2009scheduled time point(s) to ensure rapid transport to the laboratory aftercollection.7.2.3 Of the 4 labels prepared for each sample, one label is placed onto thefreshly collected sample CPT, and a second label is given to the researchnurse to place into the patient record sheet.7.2.4 The sample is transported at room temperature (RT) in a double containerfrom the clinical collection site to the sample processing laboratory. ABatch Record is to be started for each specimen (Appendix 1).7.2.5 The blood sample is mixed by inverting the tube gently 5 to 8 times andthen centrifuging at 1,500 x g for 30 min at 18°C to 25°C, without thebrake.7.2.6 Place the third identical label onto the laboratory tracking sheet, and placethe fourth label onto a sterile 15-mL conical tube.7.2.7 After centrifugation, using a 3-mL Falcon transfer pipette, carefullyremove two-thirds of the upper plasma layer and discard in biologicalwaste. Use care not to disrupt the underlying material. Change pipettetips and transfer the whitish layer that contains the PBMCs into the labeled15-mL conical tube. Discard the remaining liquid and Vacutainer CPT inthe appropriate biohazardous waste container(s).7.2.8 Using a pipette, slowly add Plasma-Lyte A USP to the PBMCs in the15-mL tube to bring the total volume to 14 mL; cap, then mix by gentleinversion 5 to 8 times.7.2.9 Centrifuge the sample at 430 x g for 10 min at 18°C to 25°C, without thebrake.7.2.10 Using a sterile pipette, aspirate as much supernatant as possible withoutdisturbing the cell pellet. Discard the supernatant into biohazardous liquidwaste.7.2.11 Add 6 mL of Plasma-Lyte A USP to the tube, and resuspend the cell pelletby gently flicking the bottom of the tube with the index finger, and thengently pipetting up and down 5 times using a 5-mL pipette.7.2.12 Immediately after resuspending the cell pellet, transfer 20 µL of sampleinto a microtube containing 60 µL Plasma-Lyte A USP and 20 µL of0.4% Trypan Blue, and set aside for a cell count.

NCTVL Standard Operating Procedure (SOP)Title: PBMC Specimen Processing for PAR Immunoassay Page 6 of 12Doc. #: SOP340521 Revision: RETIRED Effective Date: 7/24/20097.2.13 Centrifuge the remaining 5.98-mL cell suspension at 430 x g for 10 min at18°C to 25°C, without the brake. While centrifuging, after the TrypanBlue sample has incubated for 2 to 5 min, determine the total and viablecell count using a hemacytometer.7.2.14 Record the following information in Section 2 of the Batch Record(Appendix 1):• Total cell count in each hemacytometer square that was counted.• Total viable cell count in each hemacytometer square that wascounted.• Calculated viable cell concentration in the 6.0-mL cell suspension.• Viable cell yield in the remaining 5.98-mL cell suspension.7.2.14.1 Based on the total viable cell yield, proceed as follows:a. If cell yield is ≥ 6.5 x10 6 , then calculate the volume to add in orderto make the cell concentration of PBMC suspension equal to 3 x10 6 cells/mL.b. If cell yield is < 6.5 x 10 6 , then calculate the volume required tomake the suspension equal to 1.5 x 10 6 cells/mL.7.2.15 Without disturbing the cell pellet, remove supernatant and discard in anappropriate biohazardous waste container.7.2.16 Add to the cell pellet the volume of Plasma-Lyte A USP calculated in SOPStep 7.2.14 to yield a cell suspension containing 3 x 10 6 , or if required1.5 x 10 6 , viable PBMCs per mL. Resuspend the cell pellet by gentlyflicking the bottom of the tube with the index finger and then gentlypipetting up and down 5 times using a 2-mL pipette for volumes of 2.5 mLor less, or a 5-mL pipette for volumes greater than 2.5 mL.7.2.17 Based on the PBMC cell suspension concentration, proceed as follows:a. For the 3 x 10 6 cells/mL suspension, aliquot the PBMC suspensioninto 2.0-mL Sarstedt tubes using 1.0-mL aliquots until the remainingvolume of cell suspension is less than 1.0 mL. Record the number of2.0-mL Sarstedt tubes that have been prepared in the Batch Record(Appendix 1). Discard the remaining cells as biohazardous waste.b. For the 1.5 x 10 6 cells/mL cell suspension, aliquot the PBMCsuspension into 1.5-mL Sarstedt tubes using 1.0 mL aliquots untilthe remaining volume of cell suspension is less than 1.0 mL. Recordthe number of 1.5-mL Sarstedt tubes that have been prepared in the

NCTVL Standard Operating Procedure (SOP)Title: PBMC Specimen Processing for PAR Immunoassay Page 7 of 12Doc. #: SOP340521 Revision: RETIRED Effective Date: 7/24/2009Batch Record (Appendix 1). Pipette the residual volume of cellsuspension remaining in the tube into a 1.5-mL Sarstedt tube, notingthe actually volume. Write “partial” on the top of the tube with ablack Sharpie. Record the preparation of the single residual-volumevial in Section 3 of the Batch Record (Appendix 1).• Note: Based on the initial cell yield determined in Section 7.2.14,either a 2.0-mL (3.0 x 10 6 cells/mL) or 1.5-mL (1.5 x 10 6 cells/mL)Sarstedt tube is used to differentiate the final concentration of thecell suspension.7.2.18 Centrifuge the Sarstedt tubes in Sorvall Fresco centrifuge at 12,000 rpm(12,000 x g) for 10 min at 4°C to 10°C.7.2.19 Remove and discard as much supernatant as possible without disturbingthe cell pellet. Place supernatant into biohazardous waste.7.2.20 Place appropriate labels onto each Sarstedt tube, indicating specimen type,ID, and date.7.2.21 Snap-freeze the Sarstedt tube containing the PBMC cell pellets usingliquid nitrogen or a dry ice/ethanol bath.7.2.22 Store the frozen PBMC samples at -80°C until analysis.7.2.23 Complete Batch Record (Appendix 1), review, obtain required signatures,and file.7.2.24 All activities with these specimens should be tracked and noted in thespecimen file (i.e., extraction and analysis, shipment to another site, etc).7.3 PBMC Cell Pellet Extraction for PAR Immunoassay7.3.1 Add 300 µL of Cell Extraction Buffer (CEB) containing 1X proteaseinhibitor (PI) cocktail and 1 mM PMSF to fresh or frozen cell pellet in a2.0-mL Sarstedt tube containing 3.0 x 10 6 cells. The appropriate ratio ofbuffer to cell number is 100 µL of CEB per 1.0 x 10 6 cells.7.3.2 Vortex tube for 3 to 5 sec at medium speed (setting 5-6 on VortexGenie 2). Ensure the cell pellet is dislodged and mixing gently in theCEB.

NCTVL Standard Operating Procedure (SOP)Title: PBMC Specimen Processing for PAR Immunoassay Page 9 of 12Doc. #: SOP340521 Revision: RETIRED Effective Date: 7/24/2009APPENDIX 1: BATCH RECORD PAGE 1Each experimental run must be accompanied by a completed Batch Record with each page datedand initialed.Lab Technician (Certification number): ( )Date: __________________Reviewer/Supervisor:Date:Lot numbers:Vacutainer CPT Product # and Lot #:______________________________________________Plasma-Lyte A USP Lot # and expiration date: ______________________________________15-mL polypropylene tubes (circle one):Fischer14-959-49BBecton Dickinson 352097 or 352095Other: _____________Sarstedt tube Lot #:___________________________________________________________Trypan Blue Lot #_________________________ Dilution Vials______________________Serial numbers of equipment:P-100 Pipetman: P-1000 Pipetman:Processing Records:1. Preparation of ReagentsName Stock Working SolutionCell ExtractionBuffer (CEB)Protease Inhibitor(PI) CocktailPMSFPre-made 1X bufferRemove sufficient volume for number ofsamples to be processed at 500 µL/20 mgtissue.25X: 1 tablet in 2 mL ddH 2 O 1X: 40 µL 25X Stock in 1 mL CEB100 mM Manufacturer’s StockSolution1 mM: 10 µL of 100 mM Stock in 1 mLCEB (add PMSF immediately before use)INITIALSDATE:

NCTVL Standard Operating Procedure (SOP)Title: PBMC Specimen Processing for PAR Immunoassay Page 10 of 12Doc. #: SOP340521 Revision: RETIRED Effective Date: 7/24/2009BATCH RECORD PAGE 2NOTE:Record times using military time (24-h designation). Create a new BatchRecord for each patient and each patient sample.2. Isolation of PBMCsSample ID: ___________________________________________________________________Study Project ID: ______________________________________________________________Blood Volume ______________________ Time of Venous Blood Draw: ________________Time Lab Processing Begins: ____________________________________________________Time of PBMC Transfer into Plasma-Lyte A USP: ____________________________________Time of Cell Counts in Hemacytometer: ____________________________________________Record the following data:Total cell counts in each hemacytometer square counted: ____ ____ ____ ____Viable cell counts in each hemacytometer square counted: ____ ____ ____ ____Hemacytometer dilution factor: _____________________________________________Calculated viable cell concentration in suspension: ______________________________Viable cell yield remaining in the 5.98-mL cell suspension: ________________________Volume used to resuspend cell pellet to 3 x 10 6 viable PBMCs per mL: ______________INITIALSDATE:

NCTVL Standard Operating Procedure (SOP)Title: PBMC Specimen Processing for PAR Immunoassay Page 11 of 12Doc. #: SOP340521 Revision: RETIRED Effective Date: 7/24/2009BATCH RECORD PAGE 3Sample ID from Section 2 of Batch Record:3. AliquotingCHOOSE ONE of the following actions by checking the appropriate box: For PBMC yields of ≥ 6.5 x 10 6 cells, diluted to 3.0 x 10 6 cells/mL:Number of 2.0-mL Sarstedt tubes with 1.0 mL cell suspension:Location: For PBMC yields of < 6.5 x 10 6 cells, diluted to 1.5 x 10 6 cells/mL:Number of 1.5-mL Sarstedt tubes with 1.0 mL cell suspension:Location:Volume of “partial” in 1.5-mL Sarstedt tube with

NCTVL Standard Operating Procedure (SOP)Title: PBMC Specimen Processing for PAR Immunoassay Page 12 of 12Doc. #: SOP340521 Revision: RETIRED Effective Date: 7/24/2009BATCH RECORD PAGE 44. Extraction of PBMC Cell Pellet for PAR ImmunoassayNumber of PBMC vials: _____________Prepare CEB ( PBMC vials + 2) x 300 µL = µLAdd 25X PI stock _____ µL Add 100X (100 mM) PMSF _______µLAdd 300 µL or µL of CEB into each sample vial.Lyse cell pellet on ice for 30 min: Start time: ________ Stop time: ________Add 15 µL or µL of 20% SDS into each sample before boiling cell lysate for 5 min at100°C.Date of Freezing of Extract: ___________ Date of Thawing of Extract: ___________Number123456789101112131415SampleName/IDNumber ofCells/vial(x10 6 )Add CEB(µL)Add 20%SDS (µL)PBMCConcentration(10 6 /mL)INITIALSDATE: